1. Field of the Invention
This invention relates to recombinant DNA molecules and their use in producing terminal deoxynucleotidyl transferase-like polypeptides. More particularly, the invention relates to recombinant DNA molecules expressed in appropriate host organisms. The recombinant DNA molecules disclosed herein are characterized by containing DNA sequences that code for polypeptides having an immunological or biological activity of human terminal transferase. As will be appreciated from the disclosure to follow, the recombinant DNA molecules of this invention may be used in the production of polypeptides useful for production of active enzymes for modification of DNA molecules and in production of antigen to generate diagnostic and analytical reagents.
2. Prior Disclosure
All publications or patents mentioned in this specification are herein incorporated by reference.
In this application the words "terminal deoxynucleotidyl transferase" refers to a protein originally isolated from calf thymus gland and having the unique property of being able to catalyze the polymerization of deoxynucleoside triphosphates in the presence of a suitable initiator molecule, also being made of DNA monomers. In the chemical literature the molecules produced might be called "block copolymers" or "graft copolymers". The full name referred to above is also abbreviated to "terminal transferase" or "TdT" for convenience, and may also be referred to by its accepted classification by the International Committee on Enzyme Nomenclature as EC 2.7.7.31. The reaction carried out by this enzyme can be summarized in the chemical formulation:
n dNTP+initiator.fwdarw.initiator-(dNMP)n+n pyrophosphate where dNTP stands for deoxynucleoside triphosphate, initiator is a short piece of DNA molecule containing at least 3 monomer residues and a free 3'--OH, and pyrophosphate is a molecule liberated from the activated form of the nucleotide monomer (the dNTP). The N is dNTP refers generally to a heterocyclic base, usually adenine, guanine, cytosine, or thymine, but other derivatives of these bases may also be in this reaction.
The TdT enzyme activity formulated above was first described in 1960 (F. J. Bollum, 1960, J. Biol. Chem., 235, pg 18) as a side reaction present in partially purified DNA polymerase preparations from calf thymus glands. The activity was shown to polymerize deoxynucleotides onto a preformed initiator without template direction. At that time this terminal addition activity was presumed to be related to DNA polymerase as some form of active subunit (see F. J. Bollum, 1974, The Enzymes, Academic Press, New York pp 145-184). TdT was subsequently demonstrated to be a unique entity by tissue localization (L. M. S. Chang, 1974, Biochem. Biophys. Res. Comm., 44, 124-131; M. S. Coleman, J. J. Hutton and F. J. Bollum, 1974, Blood, 44:19-32). Successful purification of TdT from calf thymus glands (L. M. S. Chang and F. J. Bollum, 1971, J. Biol. Chem., 246, 909-916) and production of immuno-affinity purified rabbit antibodies to calf thyms TdT (F. J. Bollum, 1975, Proc. Nat'l. Acad. Sci., USA, 72, 4119-4122) allowed further studies on tissue localization and the demonstration that TdT protein is highly conserved in the animal world (F. J. Bollum and L. M. S. Chang, 1981, J. Biol. Chem., 256, 8767-8770). It is clear today that TdT is indeed a most rare and unusual DNA polymerase found only in pre-lymphocytes in early stages of lymphoid differentiation. The restriction of tissue localization to specific thymus and bone marrow cells and the detection of the expanded TdT-positive population occurring in accute leukemias resulted in practical rewards that were totally unpredicted. Today, immunochemical (F. J. Bollum, 1979, Blood, 54, 1203-1215) and enzymatic determinations of TdT provide the basis of diagnostic classification of human lymphoid leukemia in the pre-B and pre-T lineages (L. M. S. Chang and F. J. Bollum, Advances in Cancer Research, 1986) and differential diagnosis of myeloid leukemias. The cell biology of TdT and uses of TdT as in leukemia diagnosis has been discussed in several reviews (vide supra).
Although the publication by Landau et al. (N. R. Landau, T. P. St. John, I. L. Weissman, S. C. Wolf, A. E. Silverstone and D. Baltimore, 1984, Proc. Nat'l. Acad. Sci., USA, 81, 5836-5840), alleges to have isolated a mouse TdT cDNA plasmid, it contains no sequence information and therefore does not instruct and it therefore would not enable one of ordinary skill in the art to practice our invention. Other results such as those disclosed by R. C. Peterson, L. C. Cheung, R. J. Mattaliano, L. M. S. Chang and F. J. Bollum, 1984, Proc. Nat'l. Acad. Sci., USA, 81, 4363-4367, are limited to the finding that only 30% of the cDNA sequence claimed in this application and could not by itself produce the cDNA sequence as claimed.
Employment of TdT to modify DNA molecules by adding homopolymer tails to isolated DNA molecules (D. A. Jackson, R. H. Symons and P. Berg, 1972, Proc. Nat'l. Acad. Sci., USA, 69, 2904-2909); P. E. Lobban and A. D. Kaiser, 1973, J. Mol. Biol., 78:453-469) provided the first method for producing test tube recombinant DNAs and remains as an important tool in recombinant DNA technology. There are now many practical uses for this enzyme (F. J. Bollum, 1981, TIBS, 6, 41-43). The invention described provides a new way for producing this rare material in rapid ways and with new versatility.